DNA Damage-induced Down-regulation of Human Cdc25C and Cdc2 Is Mediated by Cooperation between p53 and Maintenance DNA (Cytosine-5) Methyltransferase 1

Gerald Le Gac,Pierre-Olivier Estève,Claude Ferec,Sriharsa Pradhan

doi:10.1074/jbc.m603724200

Gerald Le Gac, Pierre-Olivier Estève + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m603724200

Copy DOI

Abstract

The Cdc25C phosphatase mediates cellular entry into mitosis in mammalian cells. Cdc25C activates Cdc2 for entry into mitosis by dephosphorylating Thr and Tyr at the site of inhibitory phosphorylation. The Cdc25C gene contains tumor suppressor p53 binding sites and is demonstrated to contribute to the p53-dependent cell cycle arrest upon DNA damage. Here we show that both Cdc25C and Cdc2 were down-regulated in wild-type HCT116 cells but not in p53-null, DNMT1-null or DNMT1and DNMT3b-null cells, upon p53 stabilization following doxorubicin-mediated DNA damage. Furthermore, zebularine, a drug that selectively traps and depletes nuclear DNMT1 and DNMT3b, relieved p53-mediated repression of endogenous Cdc25C and Cdc2. Methylation analysis of the Cdc25C and Cdc2 promoter displayed internal CG methylation proximal to the p53 binding site upon DNA damage in a p53-dependent manner. Chromatin immunoprecipitation of doxorubicin treated wild-type HCT116 cells showed the presence of DNMT1, p53, H3K9me2, and the transcriptional repressor HDAC1 on the Cdc25C and Cdc2 promoters, suggesting their involvement as repressive complexes in Cdc25C and Cdc2 gene silencing. Thus, the general mechanism of p53-mediated gene repression may involve recruitment of other repressive factors.

Highlights

Because the majority of CpG dinucleotides in the mammalian genome are methylated and methylation often dictates the transcriptional status of a gene
It has been demonstrated that DNA methylation is correlated with transcriptional inactivation of a gene and the reverse is true for gene activation, recently it was reported that histone modification greatly influences the transcriptional status of a gene [3]
DNMTs enforce gene silencing directly by DNA methylation, they act as a platform for recruitment of transcriptional repressor complexes for gene silencing through their N-terminal regions [7]

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Parental HCT116 (colorectal carcinoma) cells were purchased from the American Type Culture Collection. Western Blot Analysis—Subconfluent cultures of HCT116 cells were washed with 1ϫ PBS, and lysed at 4 °C for 20 min with wash buffer (50 mM Tris-Cl pH 8.0, 150 mM NaCl, 0.02% sodium azide, 100 ␮g/ml phenylmethylsulfonyl fluoride, and 1% Nonidet P-40) supplemented with a protease inhibitor mixture (Sigma). Real-time PCR efficiency (E) of each of the target and reference gene (G6PDH) transcript was investigated from 40-ng to 64-pg cDNA dilutions (Table 1). Luciferase Activity Assays—The pGL3 reporter plasmids were transiently transfected into wild-type and mutant HCT116 cells (cultured in 6-well plates) using the FuGENE method according to the manufacturer’s recommendations (Roche Applied Science) and using a 4.5:1.2 transfection reagent (␮l)/DNA ratio (␮g). Gene-specific primers flanking p53-binding site of Cdc25C and Cdc promoter amplified the digested DNAs. Chromatin Immunoprecipitation Assay—HCT116 cells were grown on 150-mm dishes and treated with 1 ␮M of doxorubicin.

RESULTS

DNA is believed to facilitate

Findings

DISCUSSION